Aldosterone and its principal receptor, the mineralocorticoid receptor (MR), play essential roles in mammalian physiology, and pathophysiology, particularly due to their effects on ion transport in the kidney tubules. We previously identified SGK1 as an aldosterone/MR-regulated gene in kidney collecting duct (CD), and its functional significance in mediating aldosterone effects is now well established. However, SGK1 is not the sole mediator of aldosterone effects, and hence, we undertook a screen to identify additional aldosterone-regulated genes in kidney CD cells. One of the early induced genes, GILZ (glucocorticoid-induced leucine zipper protein) had several features suggesting that it played an important role in mediating aldosterone actions in CD, due to its inhibitory effect on the extracellular signal-regulated kinase (ERK1/2), which itself has been implicated in inhibiting epithelial Na+ transport. These findings have suggested novel organizing hypotheses that direct our future efforts: 1) GILZ is a scaffolding protein, which exerts its inhibitory effect on ERK1/2 by binding to the upstream kinase, Raf and displacing its activator, Ras; 2) GILZ acts to regulate the epithelial Na+ channel (ENaC) by decreasing its plasma membrane expression; 3) GILZ and SGK1 act through converging pathways to regulate ENaC coordinately. Based on these hypotheses, we have the following specific aims for the present research: Aim 1: Determine the mechanistic basis of GILZ inhibition of ERK. We will use a combination of in vitro and cell culture approaches to identify the direct target(s) of GILZ action. We will examine GILZ effects on the subcellular localization of signaling components, and whether they colocalize with ENaC. Aim 2: Determine the mechanistic basis and physiological relevance of GILZ and ERK regulation of ENaC function. We will examine the effect of wild type and mutant GILZ on ENaC-mediated Na+ currents, and compare ENaC regulation with effects on other ERK-mediated signals. Aim 3: Examine the effect of ERK1/2 and GILZ on ENaC trafficking, open probability and processing. This work will provide novel insight into the mechanism of aldosterone signaling, and more broadly into how signaling pathways are modulated in the control of ion transporter trafficking and activity. Determining how specific effects on ENaC can be selectively elicited by pleiotropic pathways may provide the basis for identification of specific inhibitors that impact selectively on ion transport processes.